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16th SEPTEMBER 2014
Salão Nobre, ICBAS-UP
Rua de Jorge Viterbo Ferreira n.º 228
Porto,
PORTUGAL
ANIMAL SCIENCE
DOCTORAL
PROGRAMME
- I WORKSHOP
ANIMAL SCIENCE DOCTORAL PROGRAMME
- I WORKSHOP
16th
September 2014
Organizers
António Mira Fonseca, REQUIMTE & ICBAS
Luísa Maria Pinheiro Valente, CIIMAR & ICBAS
Ana Rita Cabrita, REQUIMTE & ICBAS
Secretariat
Graciliana Lopes, CIIMAR
Vera Sousa, ICBAS
Inês Campos, ICBAS & CIIMAR
Conference Chairman
António Manuel de Sousa Pereira, ICBAS
Ana Rita Cabrita, REQUIMTE & ICBAS
Jorge Dias, Sparos
AGENDA
14:00 – Opening Session: Chairperson António Manuel de Sousa Pereira, ICBAS-UP Director
Baltazar Manuel Romão de Castro, REQUIMTE President
Vítor Manuel Oliveira Vasconcelos, CIIMAR Director
14:30 – Presentation of the new Doctoral ANIMAL SCIENCE Programme in an industrial setting
funded by FCT: SANFEED - Sustainable Animal Nutrition and Feeding
António Mira Fonseca, REQUIMTE & ICBAS-UP and Luísa MP Valente, CIIMAR
& ICBAS-UP
Session I: Chairperson Ana Rita Cabrita, REQUIMTE & ICBAS
15:00 – Plenary session: European Animal Feed Market – New trends and perspectives
Emídio Gomes, Portugal North Autorithy & ICBAS-UP
15:30 – Mediterranean Grain Legumes: Chemical Composition and Bioactive Compounds
Sara C. Q. Magalhães, REQUIMTE & ICBAS-UP PhD student
15:45 – Modelling the temperature and pH decline early post-mortem of the beef carcasses
Cristina Xavier, CIMO & ICBAS-UP PhD student
16:00 – Effects of salinity challenge on ion regulation in estuarine migrating sea lamprey, Petromyzon
marinus
Diogo Martins, CIIMAR & ICBAS-UP PhD student
16:15 – Young of the Year (YOY) flounder condition and relationship with habitat features in the Lima
estuary (NW Portugal)
Cláudia Mendes, ICBAS-UP PhD student
16:30 – Coffee Break
Session II: Chairperson Jorge Dias, Sparos
17:00 – Plenary session: Future directions in Fish Nutrition
Sadasivam Kaushik, INRA
17:30 - Use of probiotics intercropped with plant protein diets and their influence on the growth
performance and immunological status of Senegalese sole, Solea senegalensis
Sónia Batista, CIIMAR & ICBAS-UP PhD student
17:45 – Development and establishment of an effective and reproducible infection model of
Tenacibaculum maritimum against Senegalese sole (Solea senegalensis)
Mahmoud Mabrok CIMAR & ICBAS-UP PhD student
18:00 - Effect of seaweed supplementation on growth performance, immune and oxidative stress
responses in gilthead seabream (Sparus aurata)
Augusto C. Queiroz, ICBAS-UP PhD student
18:15 - Protein hydrolysates affect protein accretion and the regulation of muscle growth in
Senegalese sole larvae
Paula Canada, CIIMAR/CCMAR & ICBAS-UP PhD student
18:30 – Discussion: Chairperson Luísa MP Valente, CIIMAR & ICBAS-UP
ORAL COMMUNICATIONS
THE COMPOUND FEED INDUSTRY IN THE EU LIVESTOCK ECONOMY
Emídio Gomes
ICBAS-CCDRN
The compound feed industry has become capital intensive in recent years and makes use
of a very high level of technology. Advanced methods are used to formulate feeds
according to the demands of the livestock farmer – which reflects final consumers’ demand – and to control the raw materials used, the manufacturing process and the
quality of the finished feeds. The compound feed industry is subject to a complex body
of both EU and national legislation affecting almost every part of its operation. This
legislation is designed to ensure that feeds are of high quality and are safe for both
livestock and consumers.
Within the EU-27, about 470 mio. t of feedingstuffs are consumed by livestock each
year. Out of this quantity, 230 mio. t mostly are roughages grown and used on the farm
of origin. The balance, i.e. 240 mio. t of feed, includes cereals grown and used on the
farm of origin (53 mio. t) and feed purchased by livestock producers to supplement their
own feed resources (either feed materials or compound feed).
In 2012, 153.5 mio. t of compound feed were produced by EU compounders, accounting for 82% of all purchased feedingstuffs. Purchases of compound feed amounted, in 2011,
to €51 billion. Compound feed are manufactured from a mixture of raw materials
designed to achieve pre-determined performance objectives among animals. These raw
materials are obtained from a wide variety of sources. Hence, the industry provides a
major market for EU cereals, oilseeds and pulses. Some raw materials are obtained from
the co-products of the food industry. Other important ingredients which cannot be grown
in sufficient quantity in the EU are imported from third countries. These diverse sources
of raw material supplies are an important factor in the industry's ability to manufacture
feeds of both high quality and at competitive prices for livestock farmers.
MEDITERRANEAN GRAIN LEGUMES: CHEMICAL COMPOSITION AND
BIOACTIVE COMPOUNDS
Sara C. Q. Magalhães*1, Ana R. J. Cabrita1, Marcos Taveira2, Patrícia Valentão2, Paula
B. Andrade2, António J. M. Fonseca1
1REQUIMTE/ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira, 4050-313 Porto, Portugal. 2REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, 4050-313 Porto, Portugal.
E-mail: ajfonseca@icbas.up.pt
Introduction
Grain legumes (GL) are crops of the botanical family Fabaceae grown for food and feed.
Their recognized high protein content is the reason why they are dubbed “poor man’s
meat” in low-income groups in developing countries (Tharanathan and Mahadevamma,
2003). In animal nutrition, GL also constitute appealing economical and sustainable
alternatives to the protein sources commonly used (for instance soybean meal; e.g.
Jezierny et al., 2010). Mediterranean countries present suitable edaphoclimatic
conditions for GL growth and measures towards increasing GL production have already
been purposed in the European Union. Beyond proteins, legume seeds are great sources
of energy and fiber (Jezierny et al., 2010) also containing non-nutrient bioactive
compounds that may exert positive, negative or both effects in those who ingest them
(Champ, 2002). For instance, while carotenoids and phenolic compounds confer antioxidant properties to GL, other metabolites such as oxalates, enzyme inhibitors or
alkaloids may decrease nutrients’ availability and digestibility in the gastrointestinal
tract. Therefore, it seems imperative to determine in detail the nutritive value of GL
available to better include these ingredients in humans’ and animals’ diets. The aim of
this work was to characterize several Mediterranean GL varieties regarding nutritional
and bioactive properties. Whenever possible, Portuguese (PT) varieties were compared
with those from foreign (F) countries.
Species chosen and analysis
Grain legumes studied included Kabuli (beige; n=5) and Desi (dark; n=1) chickpea
(Cicer arietinum), field pea (Pisum sativum; n=21), faba bean (Vicia faba var. minor; n=10), white (n=5), narrow-leafed (n=2) and yellow (n=5) lupins (Lupinus albus, L.
angustifolius and L. luteus, respectively), chickling vetch (Lathyrus cicero; n=1) and
common vetch (Vicia sativa; n=1) which were provided by several companies from
Portugal, Spain, France, Italy and Poland. Fourteen varieties belonged to the Portuguese
Catalog of Varieties. Proximate composition was determined in all varieties as described
by Cabrita et al. (2011). Seeds were also analyzed for fatty acids profile by GC,
according to Alves et al. (2008), organic acids by HPLC-UV following Sousa et al.
(2009), carotenoids by HPLC-DAD as described by Mariutti et al. (2012) and phenolic
compounds by HPLC-DAD according to Silva et al. (2005).
Major results
Among all samples, protein content ranged between 21.0 and 42.8% DM with values above 32% belonging to lupins. Protein fraction was characterized as being highly
soluble (62.7±5.41%) in all samples. Fattest samples were chickpeas and lupins
(4.7±0.83 and 6.2±1.74% ether extract in DM, respectively) and the major fatty acids
found in all varieties were palmitic (16:0), oleic (18:1c9) and linoleic (18:2) acids that
accounted for more than 75% of total fatty acids. Within chickpeas, the dark variety,
which is suited for animal feeding, presented less fat and starch contents and higher
levels of cell-wall components than beige seeds. Starch content ranged from 27.3 to
44.6% DM in all samples, lupins being an exception. Indeed, lupins lacked starch but
presented increased amount of non-starch polysaccharides (17-29% DM) comparatively
to the other samples. Major differences between PT and F varieties were observed in
beige chickpeas; PT seeds presented (DM basis), in average, less 4.5 percent points (pp)
of protein and more 1.4 pp of fat and 5.4 pp of starch, relatively to F ones. Also, PT faba
beans and white lupins had lower protein content, while field peas and white lupins
showed similar values between both groups.
Among all varieties, only two carotenoids were identified, namely lutein and zeaxanthin.
Lutein was present in all samples and zeaxanthin only in chickpea and lupins. Of all
chickpeas, dark variety stood out from the beige ones in terms of total carotenoids content (162.3 vs. 28.7-87.6 µg g-1 DM, respectively). Indeed, dark chickpeas present
higher antioxidant activity (Segev et al., 2010), carotenoids contributing for that. Main
differences between PT and F varieties were also found in beige chickpeas, with the
formers presenting higher total carotenoids levels.
Several organic acids were identified in GL seeds (Figure 1). Citric and aconitic acids
(antioxidant agents) were common to all varieties, the former being the major compound
in all samples. Lupins presented the highest total amount of organic acids (4.0±0.43 mg
g-1 DM) and common vetch the lowest (0.5 mg g-1 DM). Among all, oxalic acid should
be highlighted once it affects calcium and magnesium metabolism and protein digestion
when ingested mainly by monogastrics (Akande et al., 2010). Results showed field peas
and faba beans to lack oxalic acid and the other species to contain between 2.0 and 7.7
mg 100 g-1. These values are considered low for human consumption (OHF, 2008) and are below those found for soybean seeds (Massey et al., 2001). PT yellow lupin
presented higher organic acids content than F ones mainly due to increased citric acid
levels.
Figure 1. HPLC-UV organic acids profile of
chickpea var. Elvar. Peaks identification:
mobile phase (MP), oxalic acid (1), cis-
aconitic acid (2), citric acid (3), malic acid (4),
trans-aconitic acid (5) and fumaric acid (6).
With the exception of chickpea samples, in which no phenolic compounds were
detected, phenolic acids and flavones were the metabolites identified in GL seeds. The
profile quietly varied between species and in some cases within varieties of the same
species. Samples with a higher content and a more detailed profile in phenolic
compounds were field peas (0.15-0.44 mg g-1 DM) and faba beans (0.30-0.41 mg g-1
DM).
Conclusions
Although for some species, PT varieties were not as proteinaceous as F ones, they all
represent good sources of protein, energy and unsaturated fatty acids for humans and
animals. The content of xantophylls, citric acid and phenols is indicative of the
antioxidant power of these seeds in biological systems. Oxalates do not constitute a
problem in any of the samples studied.
References
Akade, K.E., et al. (2010). Pak J Nutr 9:827-832. Alves, S.P., et al. (2008). J Chromatogr A 1209:212-219.
Cabrita, A.R.J., et al. (2011). J Sci Food Agric 91:1648–1652. Champ, M., M-J. (2002). Brit J Nutr
88:S307–S319. Jezierny, D., et al., (2010). Anim Feed Sci and Tech 157:111-128. Mariutti, L.R.B., et al.
(2012). Mar Drugs 10:1498-1510. Massey, L.K., et al. (2001). J Agric Food Chem 49:4262−4266. OHF,
Oxalosis & Hiperoxaluria Foundations (2008). Available at: http://www.ohf.org/docs/Oxalate2008.pdf. Segev,
A., et al. (2010). J Food Sci 75:S115-S119. Silva, B.M., et al. (2005). J. Agric. Food Chem 53:111-122.
Sousa, C., et al. (2009). J Sci Food Agric 89:1083-1089. Tharanathan, R.N. and Mahadevamma, S. (2003).
Trends Food Sci Tech 14:507-518.
MODELLING THE TEMPERATURE AND pH DECLINE EARLY POST-
MORTEM OF BEEF CARCASSES
Cristina Xavier1,2, Ursula Gonzales-Barron1, Alexandra Muller2 and Vasco Cadavez1
1CIMO Mountain Research Centre School of Agriculture, Polytechnic Institute of Braganza, Portugal 2ICBAS Institute of Biomedical Sciences Abel Salazar University of Oporto, Portugal
Abstract
The objective of this work was to model the pH and temperature decline early post-
mortem on beef carcasses and to study the effect of gender, genotype and weight class
on the pH and temperature decline patterns. A total of 24 beef animals slaughtered in a
local abattoir were sampled. pH and temperature were recorded using an OMEGA
wireless receiver/host (UWTC-REC1). The decline of pH and temperature was modelled
using one parameterisation of the exponential decay function, and its parameters were
estimated using the software R. The fitted models were used to predict pH and temperature at 1.5 h, at 3.0 h and at 24 h; the time when pH reached 6.0, and the
temperature at which pH reached 6.0. The rate parameters of the exponential decay
function for pH (KpH) and temperature (KT) were found to be independent (r=0.35,
P>0.05). The correlation between pH at 3 h and final pH (at 24 h) was very high
(r=0.930, P<0.01). The KT was influenced by the time elapsed from slaughter until the
first recording, and by the carcass weight. In opposition, those variables did not affect
the KpH. The exponential decay function was able to model the early post-mortem
decline of both pH and temperature, and the pH at 3 hours can be used as predictor of the
final pH of beef meat.
Keywords: pH, temperature, decline, meat, exponential decay
EFFECTS OF SALINITY CHALLENGE ON ION REGULATION IN
ESTUARINE MIGRATING SEA LAMPREY, PETROMYZON MARINUS.
D. Ferreira-Martins*1,2, Stephen McCormick3, J. Coimbra1,2 J. M. Wilson1
1 Laboratory of Ecophysiology CIIMAR/CIMAR University of Porto, Portugal 2Instituto de Ciências Biomédicas Abel Salazar, ICBAS, University of Porto, Portugal. 3Conte Anadromous Fish Research Center, USGS, Turner Falls MA USA.
Abstract
The sea lamprey, Petromyzon marinus, is an anadromous species in which the adults re-
enter freshwater, and migrate upstream for terminal spawning. A reduction in salinity
tolerance has been document in migrants although the underlying mechanisms have not
been characterized. The aim of this study was to determine the capacity for marine osmoregulation in late, upstream migrants by characterizing the morphological and
physiological effects of salinity challenge from a molecular perspective. Salinity was
gradually raised until a final salinity of 17.5‰. A number of relevant blood and
intestinal parameters were measured to assess ionoregulatory and biochemical changes
as well as the expression of key ion-transport related proteins by immunoblotting (IB)
[Na+/K+-ATPase (NKA), vacuolar-type H+-ATPase, carbonic anhydrase, and
Na+:K+:2Cl-contransporter]. NKA activity was also measured, in addition to oxidative
stress indicators.
The upper lethal salinity limit was determined to be > 17.5‰ in the freshwater migrating
adult lamprey. At 17.5‰ lamprey displayed a clear osmoregulatory failure, being unable
to regulate Na+ and Cl- levels with plasma and intestinal fluid approaching
environmental concentrations (osmoconforming and failure of drinking mechanism, respectively). This was accompanied by a significant drop in hematocrit (37% to 1%)
and plasma lactate concentrations indicating hemolytic anemia. Higher plasma [ALT]
indicated tissue damage that correlates with oxidative damage to liver (high lipid
peroxidation and GST activity, and lower [GSH]). No ion transport protein response to
salinity was detected in gill, intestine or kidney by immunoblotting. The only potentially
adaptive ionoregulatory response was an increase in Na+/K+-ATPase activity in mid-
intestine.
(FCT grant PTDC/MAR/98035/2008)
YOUNG OF THE YEAR (YOY) FLOUNDER CONDITION AND
RELATIONSHIP WITH HABITAT FEATURES IN THE LIMA ESTUARY (NW
PORTUGAL)
Mendes, C.*1,2, 3, Ramos, S.2, 3, Elliott, M.3, Bordalo, A.A.1,2
1 Laboratory of Hydrobiology and Ecology, Institute of Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal 2 Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Rua dos Bragas 289, 4050-123 Porto, Portugal 3 Institute of Estuarine and Coastal Studies, University of Hull, Hull HU6 7RX, UK
Abstract
In nursery areas optimal habitat characteristics, such as high prey availability and low
predation, promote enhanced fish growth and condition. Small scale variability of
environmental parameters throughout an estuarine nursery may be responsible for
differential spatial and temporal patterns in fish density and condition. The northern
coast of Portugal represents the southern distribution limit of the flounder Platichthys flesus. Environmental changes may increase the vulnerability of juvenile flounder in this
area, resulting in poorer condition affecting fish recruitment. These aspects were
investigated in the Lima estuary (northern Portugal) using spatial and temporal patterns
of YOY, flounder condition, and its relationship with habitat features. Flounder juveniles
and their main prey (macroinvertebrates) as well as environmental data were collected
monthly between May and October, in 2010 and 2013. Generalized additive models
(GAM) relating flounder condition (Fulton’s K) to habitat characteristics showed that
sediment and water characteristics shaped spatial patterns of YOY flounder condition,
with potential effects on growth, survival and recruitment to adult populations. The
improved knowledge of habitat features promoting YOY flounder condition in its
distribution limit will help prioritize areas for conservation and efficient management.
FUTURE DIRECTIONS IN FISH NUTRITION
S. J. Kaushik
UR 1067, NuMeA, INRA, 64310 Saint Pée sur Nivelle; France. E-mail: kaushik@st-pee.inra.fr
Abstract
Over the past decades we have recorded a more than thirty-fold increase in aquaculture
production. There are also previsions that show that by 2050, this growth of aquaculture
will lead to a situation where the total volume of seafood production (fisheries + aquaculture) will be above that of any individual terrestrial animal production: beef,
poultry or pork. Since capture fisheries are stable with no prospects of any foreseeable
increase, this will have to come from aquaculture. The major responsibility of the actors
of today is to make sure that that this growth in fish production is managed in a
sustainable manner. As of today, fish farming at the global level is still dominated by
freshwater species from relatively low trophic levels. But, there are clear indications that
“feed-based aquaculture” will increase with the consequence of an increasing demand
for nutritionally complete feeds. There is also great concern as regards the reliance of
fish and shrimp feeds on fish meal (FM) and fish oil (FO) derived from capture fisheries.
Although the volume of wild fishery resources diverted for feed purposes has not
increased over the past decades and there are clear indications that they have in fact declined over the past decade. Research in the area of replacement of FM and FO as
dietary sources of essential amino acids, fatty acids and micronutrients has also made
much progress in the recent years and is reflected by the significant reductions in the
inclusion of these ingredients in the commercial feeds. Ensuring the nutritional value of
farmed fish as food for man is another issue which will continue to need our attention.
The potential consequences of the use of feeds with low marine ingredients over the full
life cycle, from larvae to broodstock, of different species also will need more attention.
With the increasing contribution of aquaculture to the human food basket, the demand
for nutrients for farming fish and shrimp will rise, competing with other animal
production sectors. This growth is also subject to competition for direct or indirect land
and water use. There is a strong demand for the assessment of the environmental
footprint of different farming systems and aquaculture products. Indeed, more than in terrestrial animal production systems, interactions between aquatic animals and the
environment can have greater consequences, assessment of which at the farm or at the
global level will need novel approaches and methods. Improving the efficiency of
resource (nutrient, energy, water, land) utilization will make aquaculture sustainable,
where nutrition research has a strong role to play.
Fish nutrition research has recognized the importance of optimal nutrient and energy
supply for improving productivity in aquaculture and have made efforts to update our
knowledge on the nutrient requirements of several fish and shrimp. But, compared to
terrestrial livestock production relying on a relatively small number of species across the
world, aquaculture involves the farming of a large number of species in a wide variety of
environments and farming systems. There is a need for precision-farming approach of all aquatic organisms all through their life cycles. As a science and practice, fish nutrition
research has a great responsibility to make the sustainable development a reality, by
judiciously exploiting emerging ever novel tools and concepts.
USE OF PROBIOTICS INTERCROPPED WITH PLANT PROTEIN DIETS AND
THEIR INFLUENCE ON THE GROWTH PERFORMANCE AND
IMMUNOLOGICAL STATUS OF SENEGALESE SOLE (SOLEA
SENEGALENSIS)
Batista S.*1,2,3
, Faria, P.2,4
, Costas, B.2, Fernandes J.M.O.
3, Valente L.M.P.
1,2, Ozório
R.O.A.1,2
1 ICBAS – Instituto de Ciências Biomédicas de Abel Salazar, Universidade de Porto, Rua Jorge
Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal. 2 CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 289, 4050-123 Porto, Portugal. 3 Faculty of Biosciences and Aquaculture, University of Nordland, 8049 Bodø, Norway 4 ESAV/IPV – Escola Superior Agrária de Viseu, Quinta da Alagoa – Estrada de Nelas, Ranhados, 3500-606 Viseu, Portugal. * Presenting author: Sonia Batista, PhD student, email: sbatista@ciimar.up.pt
Abstract
In Senegalese sole (Solea senegalensis) aquaculture, infectious diseases represent a
major challenge, leading to significant economic losses (Arijo et al, 2005). According to
FAO / WHO (2001), probiotics are defined as "live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host". Previous studies
working with different fish species provided evidence on the effectiveness of various
probiotic bacteria in promoting the fish welfare (Castex et al., 2009; Hernandez et al.,
2010; Merrifield et al., 2011).
Keywords: Probiotics, immune status, plant protein sources, growth performance,
Senegalese sole
In the current study, we evaluated the effects of dietary probiotic supplementation intercropped with plant protein as fishmeal replacement on the growth performance and
immunological status in sole (Solea senegalensis). Fish were fed for 10 weeks on six
isonitrogenous and isolipidic diets (55% protein and 8% lipid, dry matter basis),
formulated to have 35% or 72% of fishmeal replacement by plant protein (PP35 or PP72)
intercropped with three probiotic supplementation (NO – unsupplemented, PRO1 and
PRO2). The probiotics were tested at a dosage recommended by the manufacturer. PRO1
was a multi-species probiotic bacteria (Bacillus sp., Pediococcus sp., Enterococcus sp.,
Lactobacillus sp.) supplemented at 1.8 × 1010 CFU kg-1 diet (CFU - colony forming unit)
and PRO2 was an autolyzed yeast (Saccharomyces cerevisiae) supplemented at 4g kg-1
diet. Zootechnical parameters were measured at start and the end of the feeding trial for
growth performance determination. Plasma was collected at 3, 17, 38 and 73 days of feeding dietary treatment for the determination of innate immune response (plasma
lysozyme and peroxidase contents, plasma alternative complement pathway activity –
ACH50) in order to detect differences between treatments as well to identify the
influence of long or short-time probiotic supplementation.
Fish with an initial body weight of 33.1 ± 0.2 g grew to a maximum final body weight of
50.6 ± 1.2 g. PRO1 groups had significantly lower weight gain and higher feed
conversion ratio (11.9 ± 1.9 and 2.2 ± 0.4, respectively) compared to NO groups (17.4 ±
1.9 and 1.5 ± 0.1 respectively). Growth performance from PP35 and PP72 groups did not
differ, suggesting that sole can cope well with diets containing high plant protein levels,
as already reported by Silva et al. (2009).
At 3 and 73 days of feeding trial, the immune parameters analysed did not present
differences among treatments, indicating that these may not be the most appropriate
sampling times to detect the influence of probiotic administration in Senegalese sole.
Plasma peroxidase content (EU mL-1) was not affected by PP level, but was significantly
changed by probiotic administration at 38 days of feeding. PRO1 groups presented
higher plasma peroxidase content (79.3 ± 18.6) comparing to PRO2 groups (40.1 ± 10.3),
but were not statistically different from NO groups (57.2 ± 14.0). Plasma lysozyme
content (EU mL-1) and ACH50 (Units ml-1) were not affected by probiotic
administration. However, fish fed PP72 diets had higher lysozyme and ACH50 levels at
38 days (1018 ± 204 and 257 ± 21, respectively) than fish fed PP35 diets (646 ± 154 and
183 ± 17, respectively). Also at 17 days, fish fed PP72 diets had higher lysozyme (345 ± 82) comparing to fish fed PP35 diets (175 ± 37).
In conclusion, the results of our study suggested that high plant protein inclusion levels
may affect Senegalese sole immune status, without affecting growth performance. Multi-
species probiotic (PRO1) was more effective in stimulating the humoral innate immune
parameters than Saccharomyces cerevisiae (PRO2). Further studies are needed to
evaluate whether dietary probiotic supplementation may induce higher resistance against
stress conditions and pathogens in sole.
Acknowledgements
S. M. G. Batista and B. Costas are supported by FCT – SFRH/BD/76668/2011 and SFRH/BPD/77210/2011, respectively. This work was also supported by PROBIOSOLEA project with the financial support of Quadro de Referência Estratégico Nacional – QREN and Programa
Operacional Regional do Norte – ON2 (Ref. no. 13551), supported by the European fund for regional development FEDER.
References
Arijo, S., Chabrillón, M., Díaz-Rosales, P., Rico, R.M., Martínez-Manzanares, E., Balebona, M.C., Toranzo,
A.E. & Moriñigo, M.A. (2005) Bacteria isolated from outbreaks affecting cultured sole, Solea senegalensis (Kaup). B. Eur. Assoc. Fish Pat., 25, 148–154.
Castex, M., Lemaire, P., Wabete, N. & Chim, L. (2009) Effect of dietary probiotic Pediococcus acidilactici on
antioxidant defences and oxidative stress status of shrimp Litopenaeus stylirostris. Aquaculture, 294, 306–313. DOI: 10.1016/j.fsi.2009.12.024.
FAO / WHO (2001) Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live
Lactic Acid Bacteria. Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and
Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria, Córdoba, Argentina.
Hernandez, L. H. H., Barrera, T. C., Mejia, J. C., Mejia, G.C., Carmen, M. C., Dosta, M., Andrade, R.L. &
Sotres, J.A.M. (2010) Effects of the commercial probiotic Lactobacillus casei on the growth, protein content of
skin mucus and stress resistance of juveniles of the Porthole livebearer Poecilopsis gracilis (Poecilidae). Aquac. Nutr., 16, 407–411. DOI: 10.1111/j.1365-2095.2009.00679.x
Merrifield, D. L., Bradley, G., Harper, G. M., Baker, R. T. M., Munn, C. B. & Davies, S. J. (2011) Assessment
of the effects of vegetative and lyophilized Pediococcus acidilactici on growth, feed utilization, intestinal
colonization and health parameters of rainbow trout (Oncorhynchus mykiss Walbaum). Aquac. Nutr., 17, 73–79. DOI: 10.1111/j.1365-2095.2009.00712.x
Silva J.M.G., Espe M., Conceição L.E.C., Dias J., Valente L.M.P. 2009. Senegalese sole juveniles (Solea
senegalensis Kaup, 1858) grow equally well on diets devoid of fish meal provided the dietary amino acids are
balanced. Aquaculture, 296, 309–317. DOI:10.1016/j.aquaculture.2009.08.031
DEVELOPMENT AND ESTABLISHMENT OF AN EFFECTIVE AND
REPRODUCIBLE INFECTON MODEL OF TENACIBACULUM MARITIMUM
AGAINST SENEGALESE SOLE (SOLEA SENEGALENSIS)
Mahmoud Mabrok1, 2,*, António Afonso1, 2, Luísa MP Valente1, 2, Benjamin Costas2
1ICBAS–UP, Instituto de Cîencias Biomedicas Abel Salazar, Universidade do Porto, Porto, Portugal. 2CIIMAR/CIMAR, Centro Interdisciplinar de Investigacão Marinha e Ambiental, Universidade do Porto, Porto, Portugal.
*Centro Interdisciplinar de Investigacão Marinha e Ambiental, Rua dos Bragas 289, 4050-123 Porto, Portugal. E-mail: dr.mahmoudmabrok@yahoo.com
Introduction
One of the most significant pathogen that threatens Senegalese sole (Solea senegalensis)
aquaculture is the Gram-negative bacterium Tenacibaculum maritimum, the main
etiological agent of marine tenacibaculosis. Like other filamentous bacteria, T.
maritimum presents many obstacles during laboratory work, resulting in a tendency for
aggregation, fluctuation and adhesion to all kind of substrates thus revealing difficulties in detection and recognition of the colonies. The colonies usually exhibit active
spreading (swarming) and polymorphism, making enumeration of the bacteria by
classical methods impossible. Nowadays, there is no evidence for establishment of an
effective challenge models as the bacterium presents some constrains during its culture.
Moreover, there is a considerable lack of knowledge regarding the mechanisms of
infection and the interactions between this particular pathogen and the host. The main
goals of this study were to optimize proper culturing conditions for T. maritimum in
order to draw standard growth curves, and to develop an effective and reproducible
challenge model using different bacterial strains.
Material and methods
Three T. maritimum strains (ACC6.1, ACC13.1 and ACC20.1) isolated by Prof. Alicia
Toranzo (Spain) from Senegalese sole in a local fish farm (Póvoa de Varzim, Portugal)
were used during experimental assays. Molecular identification of bacterial strains was
done according to (1). Several bacterial growth trials were performed using marine broth
(MB) supplemented with several chemical reagents as follows: Non-ionic surfactant
(Sigma) with different concentrations (0.1%, 1%, 10% and 100%) and cellulose
hydrolysis were used according to (2). Sodium dodecyl sulphonate was also added at
different concentrations (1%, 5% and 10%; Sigma). Another bacterial culture approach
was performed by inoculating the bacterial strains directly into MB with no other
reagents added and with strong aeration. Bacteria were cultured in marine agar (MA)
during 48h and inoculated in MB according to the above conditions at 25ºC and continuous shaking. Growth curves were obtained after recording optic density readings
(600 nm) and bacterial counting using a Helber count chamber every 2h during 72h.
One hundred and eight acclimated Senegalese sole without history on tenacibaculosis
were divided into three equal groups each of 36 fish. Each group was reared in a separate
recirculation seawater system (tª 23-25°C; 80% O2 saturation; salinity 32; and natural
photoperiod) comprised by six tanks with 6 fish per tank. Each group was inoculated
with three final concentrations of each T. maritimum strain in duplicate (2.6 × 105-7, 9.6
× 105-7, 4.8 × 105-7 for ACC6.1, ACC13.1 and ACC20.1, respectively) by a prolonged
bath method: 24 h with strong aeration. Another group of 36 fish was inoculated with
sterile saline solution under the same conditions and served as control. Experimentally
infected fish were monitored daily and mortalities recorded during 15 days. Dead fish
were sampled for bacteriological assessment. Bacterial colonies were identified using
morphological, physiological and biochemical tests according to (3) and confirmed by
conventional and nested PCR as previously described elsewhere (1).
Results and discussion
Among the different approaches employed with media supplementation, the non-ionic
surfactant with 100% concentration did not reveal bacterial fluctuation. However,
relative low growth was obtained compared to that obtained after direct inoculation in
MB and strong aeration, which was selected to study T. maritimum growth. This approach showed exponential growth for all bacterial strains, which was equivalent to
4.9 × 108, 4.8 × 108 and 4.85 × 108 cfu/ml for ACC6.1, ACC13.1 and ACC20.1,
respectively. Maximum bacterial growth was achieved after 48 h incubation period while
the lag phase started at 50 h at which the bacterial concentrations and numbers began to
decrease. The experimental infection of Senegalese sole by different strains of T.
maritimum was successfully conducted through the prolonged bath method. Most fish
died during the first week in case of ACC6.1 and ACC20.1 strains while fish exposed to
ACC13.1 extended for 15 days when exposed to the highest concentration. Different
lethal dose 50 (LD50) were obtained depending on the strain: 2.6 × 105, 9.6 × 105 and
4.8 × 105 cfu/ml for strains ACC6.1, ACC13.1 and ACC20.1, respectively. Thus,
bacterial strains ACC6.1 and ACC20.1 exhibited a great similarity in their pathogenicity
whereas ACC13.1 appears to be less virulent. Interestingly, all bacterial strains used in experimental infection belong to serotype O3. All infected fish showed skin ulcerations
mainly in the dorsal side, tail rots, red mouth and fin erosions (Fig. 1A). Internally, all
fish exhibited pale gills, friable livers and severe congestion in kidneys (Fig. 1B).
Regarding bacteriological assay, all bacterial strains were successfully isolated from
external lesions and kidneys on MA and FMM after 48 h incubation period. Samples
from external and internal (mainly kidney) injuries revealed positive results for
conventional and nested PCR. Results from the present study provide a new approach for
tenacibaculosis induction without preliminary trials for gill and skin abrasion. Hence,
these findings will be instrumental to study the pathogenicity of tenacibaculosis as well
as the host/pathogen interactions, which are not clearly understood yet.
Fig. 1. Experimentally infected Senegalese sole with Tenacibaculum maritimum
showing sever tail and fin erosions (A) and friable liver and congested kidney (B).
References
(1) Avendaño-Herrera R, Magariños B, Toranzo AE et al. (2004) Species-specific polymerase chain
reaction primer sets for the diagnosis of Tenacibaculum maritimum infection. Dis Aquat Org 72:75-
83
(2) Ramothokang TR, Drysdale GD, Bux F (2003) Isolation and cultivation of filamentous bacteria
implicated in activated sludge bulking. Water SA 29:405-410
(3) Santos Y, Pazos F, Barja JL (1999) Flexibacter maritimus, causal agent of flexibacteriosis in
marine fish. In: Oliver G (ed.) ICES identification leaflets for diseases and parasites of fish and
shellfish, No. 55. ICES, Copenhagen, p 1–6.
Fig 1A Fig 1B
EFFECT OF SEAWEED SUPPLEMENTATION ON GROWTH
PERFORMANCE, IMMUNE AND OXIDATIVE STRESS RESPONSES IN
GILTHEAD SEABREAM (SPARUS AURATA)
Queiroz, A.C.S.ab* Pereira, R.c; Domingues, Ab.; Peixoto, M.Jb; Pereira, L.F.ab;
Gonçalves, J.F.Ma; Ozório, R.O.A
ab.
a ICBAS – Abel Salazar Institute for Biomedical Sciences – University of Porto, Rua Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal. b CIIMAR – Interdisciplinary Centre of Marine and Environmental Research , Rua 9 dos Bragas, 289, 4050-123 Porto, Portugal. C ALGAplus, Lda., Travessa Alexandre da Conceição S/N, 3830-196 Ílhavo, Portugal * Presenting author
Abstract
Seaweed composition include several bioactive compounds with nutraceutical
properties. The dietary seaweed supplementation in aquafeeds may give a competitive
edge over unsupplemented fish to cope to stressful conditions under aquaculture
intensive conditions. The current study was designed to evaluate the effects of seaweed
supplementation on growth performance, immune and oxidative stress responses in gilthead seabream (Sparus aurata). Three seaweed species, each from one of the
following groups, Rhodophyta (R), Phaeophyta (P), Chlorophyta (C), and a mix of all
three (M) were supplemented to the experimental diets at 2.5% and 7.5% (R2.5%,
P2.5%, C2.5%, R7.5%, P7.5% and C7.5%, M7.5%) and tested against a control
unsupplemented group (CTRL). The M7.5% diet was supplemented with 2.5% of each
of the three seaweed. A total of 360 gilthead seabream juveniles, 31.2 ± 0.6g were
allocated in 24 rectangular tanks, 115 L capacity each, with a 4L/min water renewal, and
fed twice a day until satiation for 90 days. Growth performance was significantly
improved in all seaweed treatments, mainly daily growth index was 14.4% higher than
control. Immune response did not differ among the dietary treatments. The digestive
enzyme amylase showed higher activity in all 7.5% supplemented diets with about 2.27
times fold increase. Glutathione oxidized showed significant differences to supplemented treatments with an average 44% lower. Glutathione reductase, glutathione
s-transferase, glutathione peroxidase and catalase tended increase to diets with seaweeds.
Results indicate seaweed supplementation improved growth and fish welfare, even if not
significantly, nonetheless a favorable tendency to supplemented treatments results was
disclosed.
Keywords: Seaweed; Algae, Sparus aurata; Gilthead seabream; Nutraceutical
compounds.
Acknowledgments: This research was supported by project ALGADOURADA (31-03-05-FEP-41), funded by program PROMAR, which in turn was partially supported by the European Fisheries Fund (EEF). This research was also partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT – Foundation for Science and Technology, under the project “PEst-C/MAR/LA0015/2013.
PROTEIN HYDROLYSATES AFFECT PROTEIN ACCRETION AND THE
REGULATION OF MUSCLE GROWTH IN SENEGALESE SOLE LARVAE
Paula Canada1,2,3, Sofia Engrola3, Luís E.C. Conceição4, Rita Teodósio3, Sara Mira3,
Vera Sousa1,2, Jorge M.O. Fernandes5 and Luisa M.P. Valente1,2
1 CIIMAR/CIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas 289, 4050-123 Porto, Portugal. 2 ICBAS – Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. 3 CCMar/CIMAR - Centro de Ciências do Mar, Universidade do Algarve – Campus de Gambelas, 8005-139 Faro, Portugal. 4 SPAROS Lda., Área Empresarial de Marim, Lote C, 8700-221, Olhão, Algarve, Portugal. 5 Faculty of Biosciences and Aquaculture, University of Nordland, 8049 Bodø, Norway.
Abstract
Fish nutrition research has suggested that the partial substitution of dietary native
proteins by highly hydrolysed fish meal would result in better larval perfomances. In the
last years, this hypothesis has been studied and different experimental and commercial
hydrolysates differing in the raw material have shown remarkably different effects on
larval gut maturation and/or growth. However possible transcriptomic effects underlying
such response have not been explored so far. To our knowledge, no studies have been
published regarding the effect of feed composition and/or nutritional programming on
fish larvae metabolic capacity and muscle growth via possible epigenetic mechanisms.
In the present study, we hypothesized that a 40% substitution of native proteins by a commercial fish protein hydrolysate (HYDROL diet) delivered from mouth opening
onwards would positively influence Senegalese sole larvae capacity to utilize and
deposit protein throughout metamorphosis with possible consequences on muscle
development and long-term growth. The diet based on native proteins (INTACT) was
shown to promote a higher absorption of smaller peptides at the metamorphosis climax
(17DAH), compared to the HYDROL diet. The INTACT diet has also promoted a higher
proliferative capacity of myogenic cells at both stages, as it increased the percentage of
small fibers (<5µm). However, at 36DAH the HYDROL diet upregulated the transcript levels of Myogenin (myog), which encodes a highly conserved myogenic
regulatory factor that is involved in terminal muscle differentiation. This could possibly
indicate some attempt of compensatory growth. These results suggest that the inclusion
of highly hydrolyzed protein in the diet does not promote protein utilization or growth at
later stages of sole development.
This work was supported by FEDER thought COMPETE – Programa Operacional Factores de
Competitividade (POFC) and by FCT/MCTES (PIDDAC) under the project EPISOLE (PTDC/MAR/110547/2009). P Canada and S. Engrola are supported by grants from FCT (Portugal) SFRH/BD/82149/2011 and SFRH/BPD/49051/2008.
POSTERS
USE OF PROBIOTICS IN SOLE (SOLEA SENEGALENSIS) DIET: EFFECTS
ON NUTRITION PERFORMANCE, HOST DEFENSE, MORPHOLOGY AND
ECOLOGY OF THE DIGESTIVE TRACT
Batista S.*1,2,3, Fernandes J.M.O.3, Valente L.M.P.1,2, Ozório R.O.A.1,2
1 ICBAS – Instituto de Ciências Biomédicas de Abel Salazar, Universidade de Porto, Rua Jorge Viterbo Ferreira n.º 228, 4050-313 Porto, Portugal. 2 CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 289, 4050-123 Porto, Portugal. 3 Faculty of Biosciences and Aquaculture, University of Nordland, 8049 Bodø, Norway * Presenting author: Sonia Batista, PhD student, email: sbatista@ciimar.up.pt
Keywords: Probiotics, immune status, plant protein sources, growth performance, gene
expression, intestine histology, Senegalese sole,
Abstract
In sole (Solea senegalensis) aquaculture, infectious diseases represent a major challenge,
leading to significant economic losses. Conventionally, antibiotics are the most used products for the treatment of bacterial diseases. However, sick fish eat less food and
simultaneously pathogenic drug-resistant limit their use. Probiotic is defined as a body or
substance which contributes to the proper balance of microorganisms in the digestive
tract, promoting their immune defenses and nutritional/ physiological development.
Experiments with different fish species provided evidence of the effectiveness of various
probiotic bacteria in promoting the fish well-being, especially in situations of biotic and
abiotic stress (e.g. environmental and nutritional sub-optimal conditions).
In this project, we intend to assess the nutritional, immunological and molecular genetics
basis of the protective effects of probiotic agent in sole (Solea senegalensis) nutrition.
For that purpose we have designed 3 experiments to study the effect of probiotic use on:
a) Growth performance and food utilization; b) Immune status characterization (innate
immune parameters and gene expression); c) Intestinal morphology and ecology; and d) Protective effect after stress conditions.
The 1st experiment aimed to determine the effects of dietary probiotic supplementation
on growth, gut morphology and non-specific immune parameters in Senegalese sole
(Solea senegalensis) juveniles during a 1-month trial. Fish were fed for 1 month two
diets with 1.0 or 4.6 × 106 CFU kg-1; CFU - colony forming unit) of probiotic A
(Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets
with 3.5 or 8.6 × 105 CFU kg-1 of probiotic B (Pediococcus acidilactici), and tested
against an unsupplemented diet (control). Growth performance, as well as respiratory
burst activity (ROS), nitric oxide (NO), alternative complement pathway (ACH50),
lysozyme and peroxidase activities were not affected by the dietary treatments. Probiotic
supplementation tended to increased growth homogeneity between tanks having diet A1 the best possible alternative to decrease costs associated to size grading. Villous length
and number of goblet cells of the anterior intestine did not vary among treatments.
Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to
probiotic B, when included at the lowest level (A2 vs B2). The current study indicate that
the use of the multi-specie probiotic at 1.0 × 106 CFU kg-1 might enhance protection
against pathogen outbreak and increase nutrient absorption, whereas at the highest
concentration could reduce size dispersion among tanks. From this experiment one
poster presentation (XV International Symposium on Fish Nutrition and Feeding –
ISFNF 2012) was achieved and one paper was accepted to publication (Aquaculture
Nutrition - Doi: 10.1111/anu.12191).
The 2nd experiment aimed to evaluate: a) the effects of dietary probiotic
supplementation intercropped with the use of plant protein as fishmeal replacement on the humoral innate immune parameters and related transcript levels in sole (Solea
senegalensis); and b) histological and microflora changes in sole fed plant protein diets
intercropped with the use of probiotics. Six isoproteic and isolipidic diets (55% protein
and 8% lipid, dry matter basis) contained a plant protein level of 25% or 75% (CTRL or
PP) and one of three probiotic supplementation (NO – unsupplemented, PRO1 and
PRO2), were tested during a 73 days trial. PRO1 was a multi-species probiotic bacteria
(Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.) supplemented at 1.8
× 1010
CFU kg-1
diet) and PRO2 was autolyzed yeast (Saccharomyces cerevisiae)
supplemented at 4g kg-1 diet. Zootechnical parameters were measured at the start and the
end of the feeding trial for growth performance determination. At 73 days of feeding trial, plasma was collected for the determination of the humoral innate immune
responses (plasma lysozyme and peroxidase contents and plasma alternative complement
pathway activity – ACH50). A segment from the foregut was collected for histology
evaluation (villous length, muscular layer thickness and goblet cells). Also hindgut,
rectum and head-kidney samples were collected to determine the mRNA transcript levels
of some immune related genes (lysozymes – lyzc and lyzg; heat shock proteins – hsp90a,
hsp90b and hsp90b1; iron chelating proteins – ftm and apoa1; complement factors – c3a
and c3b; the cysteine protease casp3; and oxidative enzymes – cat and gpx). Our results
suggest that plant protein based diets may affect immune gene expression of Senegalese
sole, despite not affecting growth performance. However, in our study, diets not
supplemented with probiotic showed to have better growth performance. At the gene
expression, all tissues analyzed were significantly affected by plant protein content (hindgut - hsp90b and apoa1; rectum - casp3, gpx and cat; head-kidney - hsp90b1),
presenting PP diets lower values. Also probiotic supplementation had significantly affect
hindgut gene expression (hsp90b, hsp90b1 and gpx) and rectum gene expression
(hsp90a, ftm and apoa). Histology data are still in calculation and statistical analyse.
From this experiment two oral presentation (International Meeting on Marine Research
2014 and Aquaculture Europe 2014) were achieved and two paper are being written to
future publication.
A 3rd experiment was delineated with the purpose to determine the use of autochthonous
bacteria of sole (Solea senegalensis) intestine as a potential probiotic and their effect on
welfare and growth after a pathogen challenge. This experiment will be inserted on an
AQUAEXCEL project and will start at October 2014. There are 3 different main point differing from the other 2 experiments: 1) the use of open-water flow system; b) The use
of two potential probiotic bacteria isolated from Solea senegalensis intestine; and c)
study the probiotic effect on fish post a Challenge trial.
As preliminary conclusions, we may say that probiotic use on sole diets could affect
growth performance in an ambiguous way by increasing growth homogeneity but also
decreasing growth performance. Also duodenum histology (mucus layer thickness) was
changed with type of probiotic used during experiment. Diets with high content of
vegetables ingredients have proven to stimulate the sole innate immune response. And
we may also say that the probiotic change intestinal micro flora of Senegalese sole.
However, the beneficial effects of probiotics still remain controversial and further
studies should address the mechanism of action involved in the protection against stress conditions (nutritional, environmental, infections by pathogens).
Acknowledgements S. M. G. Batista is supported by FCT – SFRH/BD/76668/2011. This work was also supported by: a) PROBIOSOLEA project with the financial support of Quadro de Referência Estratégico Nacional – QREN and Programa Operacional Regional do Norte – ON2 (Ref. no. 13551),
supported by the European fund for regional development FEDER; and b) AQUAEXCELL project.
IDENTIFICATION OF A NOVEL CARBONIC ANHYDRASE ISOFORM IN
LARVAL LAMPREY (PETROMYZON MARINUS)
D. Ferreira-Martins*1,2, Stephen McCormick3, J. Coimbra1,2 J. M. Wilson2
1Instituto de Ciências Biomédicas Abel Salazar, ICBAS, University of Porto, Portugal. 2 Laboratory of Ecophysiology CIIMAR/CIMAR University of Porto, Portugal 3Conte Anadromous Fish Research Center, USGS, Turner Falls MA USA.
Abstract
Carbonic anhydrase (CA) plays a key role in CO2 excretion and acid-base regulation in
vertebrates. The lamprey is a basal vertebrate and to date only a single CA isoform has
been identified and characterized although multiple isoforms are found in other
vertebrate groups. Lampreys have a complex life cycle in which the larvae or
ammocoetes are benthic filter feeders which undergo a dramatic metamorphosis into
parasitic feeding juveniles. In the case of Petromyzon marinus, the post-metamorphic
juveniles migrate to the sea to commence feeding.
The aim of this study was to determine if additional branchial CA isoforms are present in P. marinus gill at different life history stages. A RT-PCR approach was taken using
degenerate and consensus sequence primers with samples from adult, ammocoete and
post-metamorphic transformer stages. A novel partial sequence was identified in
ammocoete gills and the full length sequence was obtained by RACE PCR. The new
ammocoete CA transcript is 1786bp coding for a 257 aa protein that has 67.5% aa
identity with the published CA isoform from lamprey and 56.3% and 57.4% for teleost
and mammalian CAc and CA2 isoforms, respectively. Phylogenetic analysis indicates
that the ammocoete CA isoform clusters basally within the cytosolic CA clade. Higher
transcript expression was found in ammocoete compared to transformer gill. Western
blotting using a heterologous cytosolic CA antibody with gill tissue from different
ammocoete stages and transformer indicates a pronounced shift in CA size, supporting the hypothesis of changes in branchial isoform expression during metamorphosis.
Supported by FCT PTDC/MAR/98035/2008.
USING MUSCLE CELLULARITY, TRACER STUDIES AND GENE
EXPRESSION TO UNDERSTAND THE EFFECT OF DIETARY PROTEIN
COMPOSITION ON PROTEIN DIGESTIVE CAPACITY AND MUSCLE
GROWTH
Paula Canada1,2
, Sofia Engrola3, Luís E.C. Conceição
4, Rita Teodósio
3, Nadège Richard
3,
Sara Mira3, Wilson Pinto4, Ana Filipa Lopes3, Lúcia Barriga-Negra1,2, Vera Sousa1,2,
Jorge M.O. Fernandes4, Luisa L.M. Valente1,2
1 CIIMAR/CIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas 289, 4050-123 Porto, Portugal. 2 ICBAS – Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal. 3 CCMar/CIMAR - Centro de Ciências do Mar, Universidade do Algarve – Campus de Gambelas, 8005-139 Faro, Portugal. 4 SPAROS Lda., Área Empresarial de Marim, Lote C, 8700-221, Olhão, Algarve, Portugal. 5 Faculty of Biosciences and Aquaculture, University of Nordland, 8049 Bodø, Norway.
Abstract
There is evidence that prenatal nutritional supply affects skeletal muscle development in
a variety of vertebrates, with possible long-term consequences on growth potential and
flesh quality. The amino acid (AA) imbalance is one of the causative factors for
increased catabolism and possible loss of some essential AA to energy catabolism and
other metabolic purposes. The dietary protein content and quality, may therefore,
determine fish larvae capacity to digest protein and utilize it for muscle growth. In
particular, the availability of certain amino acids seems to play a major role in
stimulating protein accretion and skeletal myofibre numbers. However, the knowledge
about the influence of early nutritional programming on muscle development and growth
is still rudimentary in fish. Even more unclear are the mechanisms underlying such
possible influence. For that reason we decided to investigate whether the dietary protein composition could
influence skeletal muscle cellularity and growth in a fish species that undergoes a
complex metamorphosis. In Senegalese sole larvae fed inert diets from mouth opening,
increased dietary protein content showed a positive effect on larval growth from 2 to 51
DAH overcoming the effect of an imbalanced AA profile. However, no major influence
was found on the larvae capacity to digest and retain protein. Although the proliferative
capacity of myogenic cells and the growth potential of Senegalese sole larvae remained
unaffected at the metamorphosis climax (19 DAH), at juvenile stage (51DAH), the
dietary protein composition did affect muscle cross-sectional area. The expression levels
of growth-related genes such as the myogenesis regulatory factors (MRFs) were studied
in an attempt to highlight the main physiological mechanisms through which the dietary protein affected muscle growth during the metamorphosis climax and at the juvenile
stage. Acknowledgments:
This work was supported by FEDER thought COMPETE – Programa Operacional Factores de Competitividade (POFC) and by FCT/MCTES (PIDDAC) under the project EPISOLE (PTDC/MAR/110547/2009). P Canada, S. Engrola and N. Richard are supported by grants from FCT (Portugal) SFRH/BD/82149/2011, SFRH/BPD/49051/2008 and SFRH/BPD/23514/2005
EFFECT OF SEAWEED SUPPLEMENTATION ON GROWTH
PERFORMANCE, IMMUNE AND OXIDATIVE STRESS RESPONSES IN
GILTHEAD SEABREAM (SPARUS AURATA)
Queiroz, A.C.S.ab* Pereira, R.c; Domingues, Ab.; Peixoto, M.Jb; Pereira, L.F.ab;
Gonçalves, J.F.Ma; Ozório, R.O.A
ab.
a ICBAS – Abel Salazar Institute for Biomedical Sciences – University of Porto, Rua Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal. b CIIMAR – Interdisciplinary Centre of Marine and Environmental Research , Rua 9 dos Bragas, 289, 4050-123 Porto, Portugal. C ALGAplus, Lda., Travessa Alexandre da Conceição S/N, 3830-196 Ílhavo, Portugal * Presenting author
Abstract
Seaweeds could be an excellent source of nutraceutical compounds, offering more
capability to cope to stressful conditions and minimizing disease outbreaks. Furthermore,
seaweed is part of the natural diet of several marine fish species, with potential
palatability properties for aquaculture diets. The current study was designed to evaluate
the effects of seaweed supplementation on growth performance, immune and oxidative stress responses in Gilthead Seabream (Sparus aurata). Three seaweed species, each
from one of the following groups, Rhodophyta (R), Phaeophyta (P) and Chlorophyta (C),
were supplemented to the experimental diets at 2.5% and 7.5% and tested against a
control diet (Ctrl) with no supplementation. In addition to six diets with seaweed in two
different levels of supplementation: R2.5%, P2.5%, C2.5%, R7.5%, P7.5% and C7.5%; a
diet Mix, supplemented at 7.5% (2.5% of each algae) was also tested. A total of 360
Gilthead Seabream fingerlings, 13.07 ± 0.13cm and 31.17 ± 0.63g were allocated in 24
rectangular tanks, 115L capacity each, with a 4L/min water renewal, and fed twice a day
until satiation. Monitored water parameters encompass: temperature, dissolved oxygen,
salinity, ammonia and nitrites, all maintained within the species wellness conditions. At
day 90 of the trial a final sampling was made. Length, weight, blood, liver and gut
samples were collected to analyze growth performance: specific growth rate and feed conversion ratio; immunologic parameters: lisozyme and peroxidase; digestives
enzymes: amylase, chimiotripsin, tripsin and lipase; oxidative stress: lipid peroxidation,
glutathione S-transferase, glutathione peroxidase, glutathione reductase, total
glutathione, glutathione oxidized, acetilcholinesterase and catalase. Growth performance
parameters showed significant difference, about all supplemented treatments than
control. In terms of the immunology, there is no signicant difference. Regarding
digestive enzymes, only amylase showed higher activity to supplemented diets.
Oxidative stress showed significant difference only to glutathione oxidized, but
glutathione reductase, glutathione s-transferase, glutathione peroxidase and catalase
tended increase to diets with seaweeds. Results indicate seaweed supplementation
improved growth and fish welfare, even if not significantly, nonetheless a favorable tendency to supplemented treatments results was disclosed.
Keywords: Seaweed; Algae, Sparus aurata; Gilthead seabream; Nutraceutical
compounds.
Acknowledgments:
This research was supported by project ALGADOURADA (31-03-05-FEP-41), funded by program PROMAR, which in turn was partially supported by the European Fisheries Fund (EEF). This research was also partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT – Foundation for Science and Technology, under the project “PEst-C/MAR/LA0015/2013.
GRAIN LEGUMES PRODUCTION UNDER RAINFED CONDITIONS IN
PORTUGAL
Sara C. Q. Magalhães*1, Ana R. J. Cabrita1, António J. M. Fonseca1
1REQUIMTE/ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira, 4050-313 Porto, Portugal. E-mail: ajfonseca@icbas.up.pt
Introduction
European Union is deficient in vegetable protein sources, being strongly dependent on
the import of protein sources (mainly oilseeds and their meals) from third countries.
Several measures have been purposed to increase grain legumes (GL) production in
European countries, namely in Portugal (Report A7-0026/2011, 2011). Despite the fact
that Portugal presents potential to produce GL, the actual area occupied by these crops is
limited due to 1) a focus of agriculture production on winter cereals, 2) no use of
legumes in rotations and 3) low yields (≤1000 kg/ha). The aim of this work was to
review the state of knowledge on the GL production in Portugal focusing on varieties of
chickpeas, field peas, faba beans and lupins, as these are important GL in Mediterranean farming systems (Carranca et al., 1999) and of interest, among others, to the feed
industry.
Edaphoclimatic conditions
Portugal is a Mediterranean country with wide precipitation variability, periodic
droughts and sudden and intensive downpours. In Entre-Douro e Minho (EDM), Beira
Litoral (BL), Trás-os-Montes (TM), and Beira Interior (BI) prevails a temperate climate
with dry or temperate summer, and in Estremadura e Ribatejo (ER), Alentejo (Alj), and
Algarve prevails a temperate climate with dry/hot summer; in a small region of Alentejo,
a cold steppe climate can also be observed. Generally, Portuguese soils are of poor
agricultural suitability as they are highly erodible, present medium to low cation exchange capacity, a pH below that considered optimal for plant growth and low levels
(~ 1%) of organic matter (REA, 1999).
Nitrogen fixation
Grain legumes establish symbiotic relationships with rhizobacteria, being able to fix N2
and to store it in root nodules. Values of N2 fixed by GL in national territory are reported
on Table 1 and are in accordance with data of other Mediterranean countries. Chickpea
was found to fix less N2 (20-60 kg N2 ha/year; Kumar and Abbo, 2001).
Table 1. Nitrogen fixation by grain legumes in Portugal.
Grain legume N2 fixed (kg/ha/year) Agricultural region Reference
Faba bean 76-125 Alentejo Carranca et al. (1999) Field pea 31-107 Alentejo Carranca et al. (1999)
White lupin >100 Ribatejo Carranca et al. (2009a) Yellow lupin 89 Trás-os-Montes Castro (1999)
Sowing season
In Mediterranean conditions, faba beans, field peas and lupins are sown in
autumn/winter while chickpea is traditionally sown in spring due to the lack of resistance
to the fungus Ascochyta rabiei (however, resistant varieties have been selected).
Portuguese studies on sowing season of GL reported that autumn sowing increased 1)
grain yield in chickpea (Figure 1), field pea and faba bean, 2) plant habit in chickpea
allowing for mechanical harvesting and 3) seeds weight in field pea (Costa Pinto et al.,
0
2000
4000
0 2000 4000
Sp
rin
g (
kg/h
a)
Autumn (kg/ha)
y = x
1990; Barroso et al., 2007). Lupins are also reported elsewhere to get higher yields in
autumn sowing (Mut et al., 2012).
Figure 1. Effect of sowing season on grain yield of chickpeas.
The results presented suggest clear advantages of autumn comparing to spring sowing.
Indeed, constrains related with delayed sowing are high temperatures/sun irradiation and
irregular/scarce rainfall, leading to shortening of growing cycle, low and irregular yields,
unsuitable plant habit and consecutively need of manual harvesting.
Multi-site yield experiments
Yield experiments in autumn sowing under rainfed conditions were already performed
with national and foreign GL varieties in different Portuguese agricultural regions. Grain
yields reported, both for national and foreign varieties, are above those traditionally
observed. With more than 2000 kg/ha, chickpea production seems also viable in TM, in addition to ER and Alj, where it is usually sown. Field pea yields above 3000 kg/ha in all
agricultural regions suggests an adaptation to a wide range of agronomic conditions.
Low grain yields of white lupins in Alj – comparatively to TM and BI – is probably
related to Alj low fertile, sandy and acidic soils, once lupins are more demanding in
terms of soil conditions. Higher yields found for bitter yellow lupins explains why
Portugal largely resort on them. However, sweet yellow lupins showed great
performances in TM.
Cereal-based rotations
In Portugal, rainfed cereals are sown in autumn/winter in N poor fields, commonly in
rotation with fallow (not recommended). Rotations with GL can greatly contribute to the development of the following cereal by restoring the N/other nutrients in the soil and
decreasing the incidence of pests/diseases in cereals and GL, constituting more economic
and sustainable farming practices. In TM, Castro (1999) found higher yields and N
levels of cereal (wheat and triticale) grain and straw when in rotation with yellow lupin,
rather than in a cereal-fallow system. Carranca et al. (2009b), in ER, reported an increase
in oat biomass when preceded by white lupin comparatively to a continuous oat-oat.
Conclusions
Several varieties of chickpea, field pea, faba bean and lupins (Portuguese or not) are
well-adapted to our edaphoclimatic conditions, capable of being sown in autumn and
grown under rainfed conditions, with final grain yields above those traditionally
observed. Grain legumes-based rotations, replacing the traditional fallow, could benefit the cereal and contribute to the development of organic and more extensive farming
systems, leading to higher farmer incomes while helping to combat human
desertification in North and Central inland, and South (Alentejo) of the country.
References
Barroso, M.R., et al. (2007). DRAPN, MADRP, Mirandela, Portugal. Carranca, C., et al. (1999). Eur J Agro
10:49-56. Carranca, C., et al. (2009a). Eur J Agro 31:183-189. Carranca, C., et al. (2009b). Eur J Agro
31:190-194. Castro, C. (1999). Degree Diss., UTAD, Portugal. Costa Pinto, L.M.R., et al. (1990). Pastagens
e Forragens 11:147-162. Kumar, J. and Abbo, S. (2001). Adv Agr 72:107-138. Mut, H.et al. (2012). J Plant
Nutr 35:1290-1302. REA, Relatório do Estado do Ambiente (1999). DGA, MADRP, Portugal. Report A7-
0026/2011 (2011). CARD, European Parliament.
LIST OF PARTICIPANTS
Aires O. Teles (FCUP/CIIMAR) Gertrude Thompson (ICAV-UP) Maria Teresa Dinis
Alexandra Müller (ICBAS-UP) Graciliana L. L. Lopes (CIIMAR) Mariana M. de Sousa (ICBAS-UP)
Alexandra Nascimento (REQUIMTE) Helena Abreu (ALGAplus) Mariana N. Barbosa (REQUIMTE/FFUP)
Alexandra Marques (CIIMAR) Henrique R. de Sousa (ICBAS-UP) Mariana S. Figueiredo
Ana Águas Hugo Oliveira (REQUIMTE/ICBAS-UP) Marina M. Casimiro (ICBAS-UP/CIIMAR)
Ana Carneiro (ICBAS-UP) Inês Costa Laranjeira Marta F. A. Pinto (FCUP)
Ana Miranda (ICBAS-UP) Inês G. Campos (ICBAS-UP/CIIMAR) Marta R. Carvalho (ICBAS-UP)
Ana Couto (FCUP/CIIMAR) Ingrid Van Dorpe (Premix Lda.) Miguel Andrade (ICBAS-UP)
Ana Gomes (CAVC) Isabel Azevedo (CIIMAR) Nuno Caminha (SORGAL)
Ana Isabel C. G. da Silva Isabel da Costa Ramos (CAVC) Paula Canada (ICBAS-UP/CIIMAR)
Ana Rita Cabrita (REQUIMTE/ICBAS-UP) Isabel Mª Lopes dos Santos (CAVC) Paula C. M. Almeida (FFUP)
Andreia Domingues (CIIMAR) Isabel Sousa Pinto (FCUP/CIIMAR) Paula C. Silva (CIIMAR/ICBAS-UP)
António Afonso (CIIMAR) Joana Carvalheiro (ICBAS-UP) Paulo A. Costa Rainha (ICBAS)
António Godinho (SORGAL) Joana Cláudia M. S. Mendes Paulo Lobo
António M. S. Pereira (ICBAS-UP) Joana Horta Soares (ICBAS-UP) Paulo R. M. Faria (SEA8 Group)
António M. Fonseca (REQUIMTE/ICBAS-UP) Joana V. Alves da Cunha (ICBAS-UP) Paulos Costa (ICBAS-UP)
Augusto C. Queiroz (ICBAS-UP/CIIMAR) João Coimbra (CIIMAR) Pedro de Jesus Pires (IPL)
Baltazar M. Romão de Castro (REQUIMTE) João Niza Ribeiro (ICBAS-UP) Rafael Henriques Ramos (UA)
Bárbara Gomes Patrício João Paulo C. Moreira Rafael Mendes Miranda
Benjamin Costas (CIIMAR) João Vasco Côrte-Real (ICBAS-UP) Raquel da Mota Magalhães (ICBAS-UP)
Bruna Lage (ICBAS-UP) John Svendeson (CIIMAR) Raquel Seabra F. dos Santos
Carlos Eduardo B. N. Morais Jorge Dias (SPAROS Lda) Renata Serradeiro (SEA8 Group)
Carole Sorreau (InVivo) José F. M. Capela (AGROS) Rita M. Mendonça (ICBAS-UP)
Catarina B. Teixeira (ICBAS-UP) José Luís F. da Costa Lima (FFUP) Roberto Branco (Beta Capital, S.A.)
Christophe Avelar C. G. Afonso (UA) José Vieira (SORGAL) Rodrigo Ozório (CIIMAR/ICBAS-UP)
Cláudia Isabel Coutinho (ICBAS-UP) Júlio Carvalheira (ICBAS-UP) Roland Bruger (InVivo NSA)
Cláudia Mendes (ICBAS-UP) Kruno Bonacic (IRTA) Rui Alves (SORGAL)
Cristina Xavier (CIMO/ICBAS-UP) Leonardo Magnoni (CIIMAR) Rui J. M. da Costa Lima (Sense Test, Lda)
Daniela Cristina Barros Silva Lourenço Ramos Pinto (ICBAS-UP) Rui Pereira (ALGAplus Lda.)
Diogo Martins (ICBAS-UP/CIIMAR) Luis Artur S. Ferreira (AGROS) Sadasivam Kaushik (INRA)
Duarte Frade (ICBAS-UP) Luís Conceição (SPAROS Lda) Sara Andreia Marques (CIBIO-UP)
Eduarda G. Neves (ICBAS-UP) Luís M. Cunha (REQUIMTE/FCUP) Sara Magalhães (REQUIMTE/ICBAS-UP)
Eduarda M. C. Neves (Sea8 Group) Luísa M. P. Valente (ICBAS-UP/CIIMAR) Sílvia Azevedo (ICBAS-UP)
Eliane P. da Silva (ICBAS-UP) Mahmoud Mabrok (ICBAS-UP/CIIMAR) Sofia Engrola (CCMAR)
Elisabete Carneiro (INVIVONSA Portugal S.A.) Manuel D. Salgueiro (AGROS) Sofia Maria Filipe Amaral (ICBAS-UP)
Elisabete Matos (SORGAL) Marcela Segundo (REQUIMTE/FFUP) Sónia Batista (ICBAS-UP/CIIMAR)
Emídio Gomes (N. Port. Authority/ICBAS-UP) Margarida Maia (REQUIMTE/ICBAS-UP) Telmo Fernandes (REQUIMTE/FFUP)
Érica Moura (ICBAS-UP) Maria A. B. G. S. Ribeiro (Sense Test, Lda) Teresa P. P. Martins (ICBAS-FCUP)
Fábio M. B. Sousa (ICBAS-UP) Maria A. Nunes (REQUIMTE/FFUP) Teresa P. de Matos Alves Leão
Filipa A. de Larangeira Lopes (ICBAS-UP) Maria A. R. Henriques (CIIMAR) Tiago Aires (SORGAL)
Filipe Soares Maria Pereira (NIOO-KNAW, Netherlands) Vera Sousa (ICBAS-UP)
Francisca Félix Melo (CIIMAR/UALG) Maria J. Peixoto (CIIMAR) Vitor M. Oliveira Vasconcelos (CIIMAR)
Francisco G. G. Carvalho (ICBAS-UP) Maria S. Leme (FCUP/CIIMAR)
PHOTO GALERY
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